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Protein misfolding and aggregation underlie the pathogenesis of many neurodegenerative diseases. In addition to chaperone-mediated refolding and proteasomal degradation, the aggresome-macroautophagy pathway has emerged as another defense mechanism for sequestration and clearance of toxic protein aggregates in cells. Previously, the 14-3-3 proteins were shown to be indispensable for the formation of aggresomes induced by mutant huntingtin proteins. In a recent study, we have determined that 14... Show moreProtein misfolding and aggregation underlie the pathogenesis of many neurodegenerative diseases. In addition to chaperone-mediated refolding and proteasomal degradation, the aggresome-macroautophagy pathway has emerged as another defense mechanism for sequestration and clearance of toxic protein aggregates in cells. Previously, the 14-3-3 proteins were shown to be indispensable for the formation of aggresomes induced by mutant huntingtin proteins. In a recent study, we have determined that 14-3-3 functions as a molecular adaptor to recruit chaperone-associated misfolded proteins to dynein motors for transport to aggresomes. This molecular complex involves a dimeric binding of 14-3-3 to both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3). As 14-3-3 has been implicated in various neurodegenerative diseases, our findings may provide mechanistic insights into its role in managing misfolded protein stress during the process of neurodegeneration. Show less

The aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts... Show moreThe aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases. Show less

14-3-3 proteins are required for hippocampal long-term potentiation and associative learning and memory.

Creator

Qiao, Haifa, Foote, Molly, Graham, Kourtney, Wu, Yuying, Zhou, Yi

Abstract/Description

14-3-3 is a family of regulatory proteins highly expressed in the brain. Previous invertebrate studies have demonstrated the importance of 14-3-3 in the regulation of synaptic functions and learning and memory. However, the in vivo role of 14-3-3 in these processes has not been determined using mammalian animal models. Here, we report the behavioral and electrophysiological characterization of a new animal model of 14-3-3 proteins. These transgenic mice, considered to be a 14-3-3 functional... Show more14-3-3 is a family of regulatory proteins highly expressed in the brain. Previous invertebrate studies have demonstrated the importance of 14-3-3 in the regulation of synaptic functions and learning and memory. However, the in vivo role of 14-3-3 in these processes has not been determined using mammalian animal models. Here, we report the behavioral and electrophysiological characterization of a new animal model of 14-3-3 proteins. These transgenic mice, considered to be a 14-3-3 functional knock-out, express a known 14-3-3 inhibitor in various brain regions of different founder lines. We identify a founder-specific impairment in hippocampal-dependent learning and memory tasks, as well as a correlated suppression in long-term synaptic plasticity of the hippocampal synapses. Moreover, hippocampal synaptic NMDA receptor levels are selectively reduced in the transgenic founder line that exhibits both behavioral and synaptic plasticity deficits. Collectively, our findings provide evidence that 14-3-3 is a positive regulator of associative learning and memory at both the behavioral and cellular level. Show less

The 14-3-3 proteins are a family of proteins that are highly expressed in the brain and particularly enriched at synapses. Evidence accumulated in the last two decades has implicated 14-3-3 proteins as an important regulator of synaptic transmission and plasticity. Here, we will review previous and more recent research that has helped us understand the roles of 14-3-3 proteins at glutamatergic synapses. A key challenge for the future is to delineate the 14-3-3-dependent molecular pathways... Show moreThe 14-3-3 proteins are a family of proteins that are highly expressed in the brain and particularly enriched at synapses. Evidence accumulated in the last two decades has implicated 14-3-3 proteins as an important regulator of synaptic transmission and plasticity. Here, we will review previous and more recent research that has helped us understand the roles of 14-3-3 proteins at glutamatergic synapses. A key challenge for the future is to delineate the 14-3-3-dependent molecular pathways involved in regulating synaptic functions. Show less

14-3-3 proteins were originally discovered as a family of proteins that are highly expressed in the brain. Through interactions with a multitude of binding partners, 14-3-3 proteins impact many aspects of brain function including neural signaling, neuronal development and neuroprotection. Although much remains to be learned and understood, 14-3-3 proteins have been implicated in a variety of neurological disorders based on evidence from both clinical and laboratory studies. Here we will... Show more14-3-3 proteins were originally discovered as a family of proteins that are highly expressed in the brain. Through interactions with a multitude of binding partners, 14-3-3 proteins impact many aspects of brain function including neural signaling, neuronal development and neuroprotection. Although much remains to be learned and understood, 14-3-3 proteins have been implicated in a variety of neurological disorders based on evidence from both clinical and laboratory studies. Here we will review previous and more recent research that has helped us understand the roles of 14-3-3 proteins in both neurodegenerative and neuropsychiatric diseases. Show less

Surface expression of voltage-gated Ca(2+) (Cav) channels is important for their function in calcium homeostasis in the physiology of excitable cells, but whether or not and how the α1 pore-forming subunits of Cav channels are trafficked to plasma membrane in the absence of the known Cav auxiliary subunits, β and α2δ, remains mysterious. Here we showed that 14-3-3 proteins promoted functional surface expression of the Cav2.2 α1B channel in transfected tsA-201 cells in the absence of any known... Show moreSurface expression of voltage-gated Ca(2+) (Cav) channels is important for their function in calcium homeostasis in the physiology of excitable cells, but whether or not and how the α1 pore-forming subunits of Cav channels are trafficked to plasma membrane in the absence of the known Cav auxiliary subunits, β and α2δ, remains mysterious. Here we showed that 14-3-3 proteins promoted functional surface expression of the Cav2.2 α1B channel in transfected tsA-201 cells in the absence of any known Cav auxiliary subunit. Both the surface to total ratio of the expressed α1B protein and the current density of voltage step-evoked Ba(2+) current were markedly suppressed by the coexpression of a 14-3-3 antagonist construct, pSCM138, but not its inactive control, pSCM174, as determined by immunofluorescence assay and whole cell voltage clamp recording, respectively. By contrast, coexpression with 14-3-3τ significantly enhanced the surface expression and current density of the Cav2.2 α1B channel. Importantly, we found that between the two previously identified 14-3-3 binding regions at the α1B C terminus, only the proximal region (amino acids 1706-1940), closer to the end of the last transmembrane domain, was retained by the endoplasmic reticulum and facilitated by 14-3-3 to traffic to plasma membrane. Additionally, we showed that the 14-3-3/Cav β subunit coregulated the surface expression of Cav2.2 channels in transfected tsA-201 cells and neurons. Altogether, our findings reveal a previously unidentified regulatory function of 14-3-3 proteins in promoting the surface expression of Cav2.2 α1B channels. Show less

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the 3'-UTR region: , which retains the endogenous 3'-UTR and , where the endogenous 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of 3'-UTR, we analyzed circadian rhythms of mice. Interestingly, mice displayed more than threefold stronger amplitude in bioluminescence rhythms than mice, and also exhibited lengthened free-running periods (∼24.0 h),... Show moreWe previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the 3'-UTR region: , which retains the endogenous 3'-UTR and , where the endogenous 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of 3'-UTR, we analyzed circadian rhythms of mice. Interestingly, mice displayed more than threefold stronger amplitude in bioluminescence rhythms than mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Analysis of the 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in mice, suggesting rhythmic overexpression of PER2 enhances expression of and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of 3'-UTR, miR-24, and PER2 in expression and core clock function. Show less

Cardiac injury induces myocardial expression of the thyroid hormone inactivating type 3 deiodinase (D3), which in turn dampens local thyroid hormone signaling. Here, we show that the D3 gene (Dio3) is a tissue-specific imprinted gene in the heart, and thus, heterozygous D3 knockout (HtzD3KO) mice constitute a model of cardiac D3 inactivation in an otherwise systemically euthyroid animal. HtzD3KO newborns have normal hearts but later develop restrictive cardiomyopathy due to cardiac-specific... Show moreCardiac injury induces myocardial expression of the thyroid hormone inactivating type 3 deiodinase (D3), which in turn dampens local thyroid hormone signaling. Here, we show that the D3 gene (Dio3) is a tissue-specific imprinted gene in the heart, and thus, heterozygous D3 knockout (HtzD3KO) mice constitute a model of cardiac D3 inactivation in an otherwise systemically euthyroid animal. HtzD3KO newborns have normal hearts but later develop restrictive cardiomyopathy due to cardiac-specific increase in thyroid hormone signaling, including myocardial fibrosis, impaired myocardial contractility, and diastolic dysfunction. In wild-type littermates, treatment with isoproterenol-induced myocardial D3 activity and an increase in the left ventricular volumes, typical of cardiac remodeling and dilatation. Remarkably, isoproterenol-treated HtzD3KO mice experienced a further decrease in left ventricular volumes with worsening of the diastolic dysfunction and the restrictive cardiomyopathy, resulting in congestive heart failure and increased mortality. These findings reveal crucial roles for Dio3 in heart function and remodeling, which may have pathophysiologic implications for human restrictive cardiomyopathy. Show less

The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington's disease. We previously found that... Show moreThe functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington's disease. We previously found that the degradation of mutated Htt with polyQ expansion (Htt103QP) depends on both ubiquitin proteasome system and autophagy. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Here, we report that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. Extended induction of Htt103QPGFP leads to the formation of a single inclusion body in wild-type yeast cells, but mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Most notably, we detected more aggregated forms of Htt103QP in sse1 Delta. mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy- mediated Htt103QP clearance, but are less critical for proteasome-dependent degradation. These findings suggest a chaperone network that facilitates inclusion body formation of misfolded proteins and the subsequent autophagic clearance. Show less

The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington's disease. We previously found that... Show moreThe functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington's disease. We previously found that the degradation of mutated Htt with polyQ expansion (Htt103QP) depends on both ubiquitin proteasome system and autophagy. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Here, we report that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. Extended induction of Htt103QP-GFP leads to the formation of a single inclusion body in wild-type yeast cells, but mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Most notably, we detected more aggregated forms of Htt103QP in sse1Δ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance, but are less critical for proteasome-dependent degradation. These findings suggest a chaperone network that facilitates inclusion body formation of misfolded proteins and the subsequent autophagic clearance. Show less

The development of novel therapies to treat chronic diabetic ulcers depends upon appropriate animal models for early stage investigation. The NONcNZO10/LtJ mouse is a new polygenic strain developed to more realistically model human metabolic syndrome and obesity-induced Type 2 diabetes; however, detailed wound healing properties have not been reported. In this report we describe a quantitative wound healing study in the NONcNZO10/LtJ mouse using a splinted excisional wound. The rate of wound... Show moreThe development of novel therapies to treat chronic diabetic ulcers depends upon appropriate animal models for early stage investigation. The NONcNZO10/LtJ mouse is a new polygenic strain developed to more realistically model human metabolic syndrome and obesity-induced Type 2 diabetes; however, detailed wound healing properties have not been reported. In this report we describe a quantitative wound healing study in the NONcNZO10/LtJ mouse using a splinted excisional wound. The rate of wound healing is compared to various controls, and is also quantified in response to topical administration of normal and mutant fibroblast growth factor-1 (FGF-1). Quantitation of re-epithelialization shows that the diabetic condition in the NONcNZO10/LtJ mouse is concomitant with a decreased rate of dermal healing. Furthermore, topical administration of a FGF-1/heparin formulation effectively accelerates re-epithelialization. A similar acceleration can also be achieved by a stabilized mutant form of FGF-1 formulated in the absence of heparin. Such accelerated rates of healing are not associated with any abnormal histology in the healed wounds. The results identify the NONcNZO10/LtJ mouse as a useful model of impaired wound healing in type II diabetes, and further, identify engineered forms of FGF-1 as a potential “second-generation” therapeutic to promote diabetic dermal wound healing. Show less

Abstract The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin... Show moreAbstract The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin desquamation and semen liquefaction, and work by a large number of investigators has elucidated pairwise and autolytic activation relationships among the KLKs with the potential for more extensive activation cascades. More recent work has asked whether functional intersection of KLKs with other types of regulatory proteases exists. Such studies show a capacity for members of the thrombostasis axis to act as broad activators of pro-KLKs. In the present report, we ask whether such functional intersection is possible between the KLKs and the members of the matrix metalloproteinase (MMP) family by evaluating the ability of the MMPs to activate pro-KLKs. The results identify MMP-20 as a broad activator of pro-KLKs, suggesting the potential for intersection of the KLK and MMP axes under pathological dysregulation of MMP-20 expression. Show less

The human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of... Show moreThe human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families. Show less

Brain-derived neurotrophic factor (BDNF) plays several prominent roles in synaptic plasticity and in learning and memory formation. Reduced BDNF levels and altered BDNF signaling have been reported in several brain diseases and behavioral disorders, which also exhibit reduced levels of AMPAr subunits. BDNF treatment acutely regulates AMPA receptor expression and function, including synaptic AMPAr subunit trafficking, and implicates several well defined signaling molecules that are required to... Show moreBrain-derived neurotrophic factor (BDNF) plays several prominent roles in synaptic plasticity and in learning and memory formation. Reduced BDNF levels and altered BDNF signaling have been reported in several brain diseases and behavioral disorders, which also exhibit reduced levels of AMPAr subunits. BDNF treatment acutely regulates AMPA receptor expression and function, including synaptic AMPAr subunit trafficking, and implicates several well defined signaling molecules that are required to elicit long term potentiation and depression (LTP and LTD, respectively). Long term encoding of synaptic events, as in long term memory formation, requires AMPAr stabilization and maintenance. However, factors regulating AMPAr stabilization in neuronal cell membranes and synaptic sites are not well characterized. In this study, we examine the effects of acute BDNF treatment on levels of AMPAr-associated scaffolding proteins and on AMPAr subunit-scaffolding protein interactions. We also examine the effects of BDNF-dependent enhanced interactions between AMPAr subunits with their specific scaffolding proteins on the accumulation of both types of proteins. Our results show that acute BDNF treatment upregulates the interactions between AMPAr subunits (GluR1 and GluR2) with their scaffold proteins SAP97 and GRIP1, respectively, leading to prolonged increased accumulation of both categories of proteins, albeit with distinct mechanisms for GluR1 and GluR2. Our findings reveal a new role for BDNF in the long term maintenance of AMPA receptor subunits and associated scaffolding proteins at synapses and further support the role of BDNF as a key regulator of synaptic consolidation. These results have potential implications for recent findings implicating BDNF and AMPAr subunits in various brain diseases and behavioral disorders. Show less

Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen.

Creator

Zhang, Yujie, Stefanovic, Branko

Abstract/Description

La ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for... Show moreLa ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for phosphorylations of other serines. Inhibition of PI3K/Akt pathway reduced the phosphorylation of LARP6, but had no effect on the S451A mutant, suggesting that PI3K/Akt pathway targets S451 and we have identified Akt as the responsible kinase. Overexpression of S451A mutant had dominant negative effect on collagen biosynthesis; drastically reduced secretion of collagen and induced hyper-modifications of collagen alpha 2 (I) polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for regulating translation and folding of collagen polypeptides. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production by human lung fibroblasts with EC50 of 150 nM. This effect can be explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of various forms of fibrosis. Show less

Date Issued

2016-03-02

Identifier

FSU_libsubv1_wos_000371176000001, 10.1038/srep22597

Format

Citation

Title

Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen.

Creator

Zhang, Yujie, Stefanovic, Branko

Abstract/Description

La ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for... Show moreLa ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for phosphorylations of other serines. Inhibition of PI3K/Akt pathway reduced the phosphorylation of LARP6, but had no effect on the S451A mutant, suggesting that PI3K/Akt pathway targets S451 and we have identified Akt as the responsible kinase. Overexpression of S451A mutant had dominant negative effect on collagen biosynthesis; drastically reduced secretion of collagen and induced hyper-modifications of collagen α2 (I) polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for regulating translation and folding of collagen polypeptides. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production by human lung fibroblasts with EC50 of 150 nM. This effect can be explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of various forms of fibrosis. Show less

Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry ... Show moreHypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD), and affinity measurements of cTnC for the thin filament in reconstituted papillary muscles to provide evidence of an allosteric mechanism in mutant cTnC that may play a role to the HCM phenotype. We showed that the D145E mutation leads to altered dynamics on a μs-ms time scale and deactivates both of the divalent cation-binding sites of the cTnC C-domain. CPMG-RD captured a low populated protein-folding conformation triggered by the Glu-145 replacement of Asp. Paradoxically, although D145E C-domain was unable to bind Ca, these changes along its backbone allowed it to attach more firmly to thin filaments than the wild-type isoform, providing evidence for an allosteric response of the Ca-binding site II in the N-domain. Our findings explain how the effects of an HCM mutation in the C-domain reflect up into the N-domain to cause an increase of Ca affinity in site II, thus opening up new insights into the HCM phenotype. Show less

Protein 3° structure symmetry is a defining feature of nearly a third of protein folds and is generally thought to result from a combination of gene duplication, fusion, and truncation events. Such events represent major replication errors, involving substantial alteration of protein 3° structure as well as causing regions of exact repeating 1° structure, both of which are generally considered deleterious to protein folding. Thus, the prevalence of symmetric protein folds is counterintuitive... Show moreProtein 3° structure symmetry is a defining feature of nearly a third of protein folds and is generally thought to result from a combination of gene duplication, fusion, and truncation events. Such events represent major replication errors, involving substantial alteration of protein 3° structure as well as causing regions of exact repeating 1° structure, both of which are generally considered deleterious to protein folding. Thus, the prevalence of symmetric protein folds is counterintuitive and suggests a specific, yet unexplained, robustness. Using a designed β-trefoil protein, we show that purely symmetric 1° structure enables utilization of alternative definitions of the critical folding nucleus in response to gross structural rearrangement. Thus, major replication errors producing 1° structure symmetry can conserve foldability. The results provide an explanation for the prevalence of symmetric protein folds, and highlight a critical role for 1° structure symmetry in protein evolution. Show less

The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca(2+)-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp... Show moreThe hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca(2+)-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca(2+) than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca(2+)-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other. Show less

The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 degrees C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a... Show moreThe hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 degrees C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 degrees C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other. Show less

Date Issued

2017-04-06

Identifier

FSU_libsubv1_wos_000398545900010, 10.1038/s41598-017-00777-6

Format

Citation

Title

Analysis of the Molecular Pathogenesis of Cardiomyopathy-Causing cTnT Mutants I79N, ΔE96, and ΔK210.

Creator

Bai, Fan, Caster, Hannah, Pinto, Jose, Kawai, Masataka

Abstract/Description

Three troponin T (TnT) mutants that cause hypertrophic, restrictive, and dilated cardiomyopathy (I79N, ΔE96, and ΔK210, respectively), were examined using the thin-filament extraction/reconstitution technique. Effects of Ca(2+), ATP, phosphate, and ADP concentrations on force and its transients were studied at 25°C. Maximal Ca(2+) tension (THC) and Ca(2+)-activatable tension (Tact), respectively, were similar among I79N, ΔE96, and WT, whereas ΔK210 led to a significantly lower THC (∼20% less)... Show moreThree troponin T (TnT) mutants that cause hypertrophic, restrictive, and dilated cardiomyopathy (I79N, ΔE96, and ΔK210, respectively), were examined using the thin-filament extraction/reconstitution technique. Effects of Ca(2+), ATP, phosphate, and ADP concentrations on force and its transients were studied at 25°C. Maximal Ca(2+) tension (THC) and Ca(2+)-activatable tension (Tact), respectively, were similar among I79N, ΔE96, and WT, whereas ΔK210 led to a significantly lower THC (∼20% less) and Tact (∼25% less) than did WT. In pCa solution containing 8 mM Pi and ionic strength adjusted to 200 mM, the Ca(2+) sensitivity (pCa50) of I79N (5.63 ± 0.02) and ΔE96 (5.60 ± 0.03) was significantly greater than that of WT (5.45 ± 0.04), but the pCa50 of ΔK210 (5.54 ± 0.04) remained similar to that of WT. Five equilibrium constants were deduced using sinusoidal analysis. All three mutants showed significantly lower K0 (ADP association constant) and larger K4 (equilibrium constant of force generation step) relative to the corresponding values for WT. I79N and ΔK210 were associated with a K2 (equilibrium constant of cross-bridge detachment step) significantly lower than that of ΔE96 and WT. These results demonstrated that at pCa 4.66, the force/cross-bridge is ∼18% less in I79N and ∼41% less in ΔK210 than that in WT. These results indicate that the molecular pathogenesis of the cardiac TnT mutation-related cardiomyopathies is different for each mutation. Show less

Neural stem cell (NSC) transplantation has been proposed to promote functional recovery after spinal cord injury. However, a detailed understanding of the mechanisms of how NSCs exert their therapeutic plasticity is lacking. We transplanted mouse NSCs into the injured spinal cord seven days after SCI, and the Basso Mouse Scale (BMS) score was performed to assess locomotor function. The anti-inflammatory effects of NSC transplantation was analyzed by immunofluorescence staining of neutrophil... Show moreNeural stem cell (NSC) transplantation has been proposed to promote functional recovery after spinal cord injury. However, a detailed understanding of the mechanisms of how NSCs exert their therapeutic plasticity is lacking. We transplanted mouse NSCs into the injured spinal cord seven days after SCI, and the Basso Mouse Scale (BMS) score was performed to assess locomotor function. The anti-inflammatory effects of NSC transplantation was analyzed by immunofluorescence staining of neutrophil and macrophages and the detection of mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-12 (IL-12). Furthermore, bone marrow-derived macrophages (BMDMs) were co-cultured with NSCs and followed by analyzing the mRNA levels of inducible nitric oxide synthase (iNOS), TNF-α, IL-1β, IL-6 and IL-10 with quantitative real-time PCR. The production of TNF-α and IL-1β by BMDMs was examined using the enzyme-linked immunosorbent assay (ELISA). Transplanted NSCs had significantly increased BMS scores (p < 0.05). Histological results showed that the grafted NSCs migrated from the injection site toward the injured area. NSCs transplantation significantly reduced the number of neutrophils and iNOS+/Mac-2+ cells at the epicenter of the injured area (p < 0.05). Meanwhile, mRNA levels of TNF-α, IL-1β, IL-6 and IL-12 in the NSCs transplantation group were significantly decreased compared to the control group. Furthermore, NSCs inhibited the iNOS expression of BMDMs and the release of inflammatory factors by macrophages in vitro (p < 0.05). These results suggest that NSC transplantation could modulate SCI-induced inflammatory responses and enhance neurological function after SCI via reducing M1 macrophage activation and infiltrating neutrophils. Thus, this study provides a new insight into the mechanisms responsible for the anti-inflammatory effect of NSC transplantation after SCI. Show less

While the influence of testosterone levels on vulnerability to affective disorders is not straightforward, research suggests this hormone may confer some degree of resiliency in men. We recently demonstrated a role for the dentate gyrus in mediating testosterone's protective effects on depressive-like behavior in gonadectomized male rats. Here, testosterone may exert its effects through androgen receptor-mediated mechanisms or via local aromatization to estradiol. Gonadectomized male rats... Show moreWhile the influence of testosterone levels on vulnerability to affective disorders is not straightforward, research suggests this hormone may confer some degree of resiliency in men. We recently demonstrated a role for the dentate gyrus in mediating testosterone's protective effects on depressive-like behavior in gonadectomized male rats. Here, testosterone may exert its effects through androgen receptor-mediated mechanisms or via local aromatization to estradiol. Gonadectomized male rats were implanted with a placebo, testosterone, or estradiol pellet, and subsequent protective anxiolytic- and antidepressant-like effects of testosterone and its aromatized metabolite, estradiol, were then investigated in the open field and sucrose preference tests, respectively. Moreover, their influence on gene expression in the hippocampus was analyzed by genome-wide complementary DNA microarray analysis. Finally, the contribution of testosterone's aromatization within the dentate gyrus was assessed by local infusion of the aromatase inhibitor fadrozole, whose efficacy was confirmed by liquid chromatography-tandem mass spectrometry. Both hormones had antidepressant-like effects associated with a substantial overlap in transcriptional regulation, particularly in synaptic plasticity- and mitogen-activated protein kinase pathway-related genes. Further, chronic aromatase inhibition within the dentate gyrus blocked the protective effects of testosterone. Both testosterone and estradiol exhibit anxiolytic- and antidepressant-like effects in gonadectomized male rats, while similarly regulating critical mediators of these behaviors, suggesting common underlying mechanisms. Accordingly, we demonstrated that testosterone's protective effects are mediated, in part, by its aromatization in the dentate gyrus. These findings thus provide further insight into a role for estradiol in mediating the protective anxiolytic- and antidepressant-like effects of testosterone. Show less

Attention And Working Memory Deficits In A Perinatal Nicotine Exposure Mouse Model.

Creator

Zhang, Lin, Spencer, Thomas J., Biederman, Joseph, Bhide, Pradeep G.

Abstract/Description

Background Cigarette smoking by pregnant women is associated with a significant increase in the risk for cognitive disorders in their children. Preclinical models confirm this risk by showing that exposure of the developing brain to nicotine produces adverse behavioral outcomes. Here we describe behavioral phenotypes resulting from perinatal nicotine exposure in a mouse model, and discuss our findings in the context of findings from previously published studies using preclinical models of... Show moreBackground Cigarette smoking by pregnant women is associated with a significant increase in the risk for cognitive disorders in their children. Preclinical models confirm this risk by showing that exposure of the developing brain to nicotine produces adverse behavioral outcomes. Here we describe behavioral phenotypes resulting from perinatal nicotine exposure in a mouse model, and discuss our findings in the context of findings from previously published studies using preclinical models of developmental nicotine exposure. Methodology/Principal findings Female C57BI/6 mice received drinking water containing nicotine (100pg/ml) + saccharin (2%) starting 3 weeks prior to breeding and continuing throughout pregnancy, and until 3 weeks postpartum. Over the same period, female mice in two control groups received drinking water containing saccharin (2%) or plain drinking water. Offspring from each group were weaned at 3-weeks of age and subjected to behavioral analyses at 3 months of age. We examined spontaneous locomotor activity, anxiety-like behavior, spatial working memory, object based attention, recognition memory and impulsive-like behavior. We found significant deficits in attention and working memory only in male mice, and no significant changes in the other behavioral phenotypes in male or female mice. Exposure to saccharin alone did not produce significant changes in either sex. Conclusion/Significance The perinatal nicotine exposure produced significant deficits in attention and working memory in a sex-dependent manner in that the male but not female offspring displayed these behaviors. These behavioral phenotypes are associated with attention deficit hyperactivity disorder (ADHD) and have been reported in other studies that used pre-or perinatal nicotine exposure. Therefore, we suggest that preclinical models of developmental nicotine exposure could be useful tools for modeling ADHD and related disorders. Show less

Date Issued

2018-05-24

Identifier

FSU_libsubv1_wos_000433084300123, 10.1371/journal.pone.0198064

Format

Citation

Title

Attention and working memory deficits in a perinatal nicotine exposure mouse model.

Creator

Zhang, Lin, Spencer, Thomas J, Biederman, Joseph, Bhide, Pradeep G

Abstract/Description

Cigarette smoking by pregnant women is associated with a significant increase in the risk for cognitive disorders in their children. Preclinical models confirm this risk by showing that exposure of the developing brain to nicotine produces adverse behavioral outcomes. Here we describe behavioral phenotypes resulting from perinatal nicotine exposure in a mouse model, and discuss our findings in the context of findings from previously published studies using preclinical models of developmental... Show moreCigarette smoking by pregnant women is associated with a significant increase in the risk for cognitive disorders in their children. Preclinical models confirm this risk by showing that exposure of the developing brain to nicotine produces adverse behavioral outcomes. Here we describe behavioral phenotypes resulting from perinatal nicotine exposure in a mouse model, and discuss our findings in the context of findings from previously published studies using preclinical models of developmental nicotine exposure. Female C57Bl/6 mice received drinking water containing nicotine (100μg/ml) + saccharin (2%) starting 3 weeks prior to breeding and continuing throughout pregnancy, and until 3 weeks postpartum. Over the same period, female mice in two control groups received drinking water containing saccharin (2%) or plain drinking water. Offspring from each group were weaned at 3-weeks of age and subjected to behavioral analyses at 3 months of age. We examined spontaneous locomotor activity, anxiety-like behavior, spatial working memory, object based attention, recognition memory and impulsive-like behavior. We found significant deficits in attention and working memory only in male mice, and no significant changes in the other behavioral phenotypes in male or female mice. Exposure to saccharin alone did not produce significant changes in either sex. The perinatal nicotine exposure produced significant deficits in attention and working memory in a sex-dependent manner in that the male but not female offspring displayed these behaviors. These behavioral phenotypes are associated with attention deficit hyperactivity disorder (ADHD) and have been reported in other studies that used pre- or perinatal nicotine exposure. Therefore, we suggest that preclinical models of developmental nicotine exposure could be useful tools for modeling ADHD and related disorders. Show less

The error-related negativity (ERN) is an electrophysiological response to errors. Individual differences in the ERN have been posited to reflect sensitivity to threat and linked with risk for anxiety disorders. Attention bias modification is a promising computerized intervention that has been shown to decrease threat biases and anxiety symptoms. In the present study, we examined the impact of a single session of attention bias modification, relative to a control task, on the neural correlates... Show moreThe error-related negativity (ERN) is an electrophysiological response to errors. Individual differences in the ERN have been posited to reflect sensitivity to threat and linked with risk for anxiety disorders. Attention bias modification is a promising computerized intervention that has been shown to decrease threat biases and anxiety symptoms. In the present study, we examined the impact of a single session of attention bias modification, relative to a control task, on the neural correlates of response monitoring, including the ERN, correct response negativity (CRN), and their difference (i.e., the ERN - CRN or ΔERN). The final sample included 60 participants who first completed a flanker task to elicit the ERN and CRN, and were then randomly assigned to attention bias modification (n=30) or a control task (n=30). After completing the attention bias modification or control task, participants completed the same flanker task to again elicit the ERN and CRN. Among participants who completed attention bias modification training, the ERN, CRN, and ΔERN decreased from the pre- to post-training assessment. In contrast, in participants who completed the control task, the CRN, ERN, and ΔERN did not differ between the pre- and post-training assessment. The presents study suggests that a single session of attention bias modification reduces neural correlates of response monitoring, including error-related brain activity. These results also support attention bias modification as a potential mechanistic-based intervention for the prevention and treatment of anxiety pathology. Show less

Aurora A Phosphorylation of YY1 during Mitosis Inactivates its DNA Binding Activity.

Creator

Alexander, Karen E, Rizkallah, Raed

Abstract/Description

Successful execution of mitotic cell division requires the tight synchronisation of numerous biochemical pathways. The underlying mechanisms that govern chromosome segregation have been thoroughly investigated. However, the mechanisms that regulate transcription factors in coordination with mitotic progression remain poorly understood. In this report, we identify the transcription factor YY1 as a novel mitotic substrate for the Aurora A kinase, a key regulator of critical mitotic events, like... Show moreSuccessful execution of mitotic cell division requires the tight synchronisation of numerous biochemical pathways. The underlying mechanisms that govern chromosome segregation have been thoroughly investigated. However, the mechanisms that regulate transcription factors in coordination with mitotic progression remain poorly understood. In this report, we identify the transcription factor YY1 as a novel mitotic substrate for the Aurora A kinase, a key regulator of critical mitotic events, like centrosome maturation and spindle formation. Using in vitro kinase assays, we show that Aurora A directly phosphorylates YY1 at serine 365 in the DNA-binding domain. Using a new phospho-specific antibody, we show that YY1 phosphorylation at serine 365 occurs during mitosis, and that this phosphorylation is significantly reduced upon inhibition of Aurora A. Furthermore, we show, using electrophoretic mobility shift and chromatin immunoprecipitation assays, that phosphorylation of YY1 at this site abolishes its DNA binding activity in vitro and in vivo. In conformity with this loss of binding activity, phosphorylated YY1 also loses its transctivation ability as demonstrated by a luciferase reporter assay. These results uncover a novel mechanism that implicates Aurora A in the mitotic inactivation of transcription factors. Show less

Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine-type proteases, characterized as an arginine-specific digestive-type protease capable of degrading a wide-variety of extracellular matrix proteins. KLK6 has been proposed to be a useful biomarker for breast and ovarian cancer prognosis, is abundantly expressed in the CNS and cerebrospinal fluid, and is intimately associated with regions of active inflammatory demyelination in multiple sclerosis (MS)... Show moreHuman kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine-type proteases, characterized as an arginine-specific digestive-type protease capable of degrading a wide-variety of extracellular matrix proteins. KLK6 has been proposed to be a useful biomarker for breast and ovarian cancer prognosis, is abundantly expressed in the CNS and cerebrospinal fluid, and is intimately associated with regions of active inflammatory demyelination in multiple sclerosis (MS) lesions. Inhibition of KLK6 results in delayed onset and reduced severity of symptoms associated with experimental autoimmune encephalomyelitis, suggesting a key effector role for this protease in CNS inflammatory disease. KLK6 has been shown to autolytically cleave internally, leading to inactivation and suggesting a negative feedback inhibition control mechanism. Alternatively, the ability of KLK6 to self-activate has also been reported, suggesting a positive feedback activation loop control mechanism. Activation of pro-KLK6 requires hydrolysis after a Lys residue; however, KLK6 exhibits 2 order of magnitude reduced affinity for hydrolysis after Lys versus Arg residues; therefore, the ability to autolytically activate has been called into question. In the present study the catalytic activity of KLK6 toward its pro-sequence and internal autolytic sequence is characterized. The results show that the ability of KLK6 to activate pro-KLK6 is essentially negligible when compared to the rate of the internal autolytic inactivation or to the ability of other proteases to activate pro-KLK6. The results thus show that the primary autolytic regulatory mechanism of KLK6 is negative feedback inhibition, and activation is likely achieved through the action of a separate protease. Show less

A balancing act: heterochromatin protein 1a and the Polycomb group coordinate their levels to silence chromatin in Drosophila..

Creator

Cabrera, Janel R, Olcese, Ursula, Horabin, Jamila I

Abstract/Description

The small non-histone protein Heterochromatin protein 1a (HP1a) plays a vital role in packaging chromatin, most notably in forming constitutive heterochromatin at the centromeres and telomeres. A second major chromatin regulating system is that of the Polycomb/trithorax groups of genes which, respectively, maintain the repressed/activated state of euchromatin. Recent analyses suggest they affect the expression of a multitude of genes, beyond the homeotics whose alteration in expression lead... Show moreThe small non-histone protein Heterochromatin protein 1a (HP1a) plays a vital role in packaging chromatin, most notably in forming constitutive heterochromatin at the centromeres and telomeres. A second major chromatin regulating system is that of the Polycomb/trithorax groups of genes which, respectively, maintain the repressed/activated state of euchromatin. Recent analyses suggest they affect the expression of a multitude of genes, beyond the homeotics whose alteration in expression lead to their initial discovery. Our data suggest that early in Drosophila development, HP1a collaborates with the Polycomb/trithorax groups of proteins to regulate gene expression and that the two chromatin systems do not act separately as convention describes. HP1a affects the levels of both the Polycomb complexes and RNA polymerase II at promoters, as assayed by chromatin immunoprecipitation analysis. Deposition of both the repressive (H3K27me3) and activating (H3K4me3) marks promoted by the Polycomb/trithorax group genes at gene promoters is affected. Additionally, depending on which parent contributes the null mutation of the HP1a gene, the levels of the H3K27me3 and H3K9me3 silencing marks at both promoters and heterochromatin are different. Changes in levels of the H3K27me3 and H3K9me3 repressive marks show a mostly reciprocal nature. The time around the mid-blastula transition, when the zygotic genome begins to be actively transcribed, appears to be a transition/decision point for setting the levels. We find that HP1a, which is normally critical for the formation of constitutive heterochromatin, also affects the generation of the epigenetic marks of the Polycomb/trithorax groups of proteins, chromatin modifiers which are key to maintaining gene expression in euchromatin. At gene promoters, deposition of both the repressive H3K27me3 and activating H3K4me3 marks of histone modifications shows a dependence on HP1a. Around the mid-blastula transition, when the zygotic genome begins to be actively transcribed, a pivotal decision for the level of silencing appears to take place. This is also when the embryo organizes its genome into heterochromatin and euchromatin. A balance between the HP1a and Polycomb group silencing systems appears to be set for the chromatin types that each system will primarily regulate. Show less

Equalizing sex chromosome expression between the sexes when they have largely differing gene content appears to be necessary, and across species, is accomplished in a variety of ways. Even in birds, where the process is less than complete, a mechanism to reduce the difference in gene dose between the sexes exists. In early development, while the dosage difference is unregulated and still in flux, it is frequently exploited by sex determination mechanisms. The Drosophila female sex... Show moreEqualizing sex chromosome expression between the sexes when they have largely differing gene content appears to be necessary, and across species, is accomplished in a variety of ways. Even in birds, where the process is less than complete, a mechanism to reduce the difference in gene dose between the sexes exists. In early development, while the dosage difference is unregulated and still in flux, it is frequently exploited by sex determination mechanisms. The Drosophila female sex determination process is one clear example, determining the sexes based on X chromosome dose. Recent data show that in Drosophila, the female sex not only reads this gene balance difference, but at the same time usurps the moment. Taking advantage of the transient default state of male dosage compensation, the sex determination master-switch Sex-lethal which resides on the X, has its expression levels enhanced before it works to correct the gene imbalance. Intriguingly, key developmental genes which could create developmental havoc if their levels were unbalanced show more exquisite regulation, suggesting nature distinguishes them and ensures their expression is kept in the desirable range. Show less

Although the Notch signaling pathway is one of the most intensely studied intracellular signaling pathways, the mechanisms by which Notch signaling regulates transcription remain incompletely understood. Here, we report that B cell leukemia/lymphoma 6 (BCL6), a transcriptional repressor, is a Notch-associated factor. BCL6 is necessary to maintain the expression of Pitx2 in the left lateral plate mesoderm during the patterning of left-right asymmetry in Xenopus embryos. For this process, BCL6... Show moreAlthough the Notch signaling pathway is one of the most intensely studied intracellular signaling pathways, the mechanisms by which Notch signaling regulates transcription remain incompletely understood. Here, we report that B cell leukemia/lymphoma 6 (BCL6), a transcriptional repressor, is a Notch-associated factor. BCL6 is necessary to maintain the expression of Pitx2 in the left lateral plate mesoderm during the patterning of left-right asymmetry in Xenopus embryos. For this process, BCL6 forms a complex with BCL6 corepressor (BCoR) on the promoters of selected Notch target genes such as enhancer of split related 1. BCL6 also inhibits the transcription of these genes by competing for the Notch1 intracellular domain, preventing the coactivator Mastermind-like1 (MAM1) from binding. These results define a mechanism restricting Notch-activated transcription to cell-type-appropriate subsets of target genes, and elucidate its relevance in vivo during left-right asymmetric development. Show less

Some species of butterflyfish have had preyed upon corals for millions of years, yet the mechanism of butterflyfish specialized coral feeding strategy remains poorly understood. Certain butterflyfish have the ability to feed on allelochemically rich soft corals, e.g. Sinularia maxima. Cytochrome P450 (CYP) is the predominant enzyme system responsible for the detoxification of dietary allelochemicals. CYP2-like and CYP3A-like content have been associated with butterflyfish that preferentially... Show moreSome species of butterflyfish have had preyed upon corals for millions of years, yet the mechanism of butterflyfish specialized coral feeding strategy remains poorly understood. Certain butterflyfish have the ability to feed on allelochemically rich soft corals, e.g. Sinularia maxima. Cytochrome P450 (CYP) is the predominant enzyme system responsible for the detoxification of dietary allelochemicals. CYP2-like and CYP3A-like content have been associated with butterflyfish that preferentially consumes allelochemically rich soft corals. To investigate the role of butterflyfish CYP2 and CYP3A enzymes in dietary preference, we conducted oral feeding experiments using homogenates of S. maxima and a toxin isolated from the coral in four species of butterflyfish with different feeding strategies. After oral exposure to the S. maxima toxin 5-episinulaptolide (5ESL), which is not normally encountered in the Hawaiian butterflyfish diet, an endemic specialist, Chaetodon multicinctus experienced 100% mortality compared to a generalist, Chaetodon auriga, which had significantly more (3-6 fold higher) CYP3A-like basal content and catalytic activity. The specialist, Chaetodon unimaculatus, which preferentially feed on S. maxima in Guam, but not in Hawaii, had 100% survival, a significant induction of 8-12 fold CYP3A-like content, and an increased ability (2-fold) to metabolize 5ESL over other species. Computer modeling data of CYP3A4 with 5ESL were consistent with microsomal transformation of 5ESL to a C15-16 epoxide from livers of C. unimaculatus. Epoxide formation correlated with CYP3A-like content, catalytic activity, induction, and NADPH-dependent metabolism of 5ESL. These results suggest a potentially important role for the CYP3A family in butterflyfish-coral diet selection through allelochemical detoxification. Show less

Background: The single-stranded RNA Flavivirus, Zika virus (ZIKV), has recently re-emerged and spread rapidly across the western hemisphere's equatorial countries, primarily through Aedes mosquito transmission. While symptoms in adult infections appear to be self-limiting and mild, severe birth defects, such as microcephaly, have been linked to infection during early pregnancy. Recently, Tang et al. (Cell Stem Cell 2016, doi: 10.1016/j.stem.2016.02.016) demonstrated that ZIKV efficiently... Show moreBackground: The single-stranded RNA Flavivirus, Zika virus (ZIKV), has recently re-emerged and spread rapidly across the western hemisphere's equatorial countries, primarily through Aedes mosquito transmission. While symptoms in adult infections appear to be self-limiting and mild, severe birth defects, such as microcephaly, have been linked to infection during early pregnancy. Recently, Tang et al. (Cell Stem Cell 2016, doi: 10.1016/j.stem.2016.02.016) demonstrated that ZIKV efficiently infects induced pluripotent stem cell (iPSC) derived human neural progenitor cells (hNPCs), resulting in cell cycle abnormalities and apoptosis. Consequently, hNPCs are a suggested ZIKV target. Methods: We analyzed the transcriptomic sequencing (RNA-seq) data (GEO: GSE78711) of ZIKV (Strain: MR766) infected hNPCs. For comparison to the ZIKV-infected hNPCs, the expression data from hNPCs infected with human cytomegalovirus (CMV) (Strain: AD169) was used (GEO: GSE35295). Utilizing a combination of Gene Ontology, database of human diseases, and pathway analysis, we generated a putative systemic model of infection supported by known molecular pathways of other highly related viruses. Results: We analyzed RNA-sequencing data for transcript expression alterations in ZIKV-infected hNPCs, and then compared them to expression patterns of iPSC-derived hNPCs infected with CMV, a virus that can also induce severe congenital neurological defects in developing fetuses. We demonstrate for the first time that many of cellular pathways correlate with clinical pathologies following ZIKV infection such as microcephaly, congenital nervous system disorders and epilepsy. Furthermore, ZIKV activates several inflammatory signals within infected hNPCs that are implicated in innate and acquired immune responses, while CMV-infected hNPCs showed limited representation of these pathways. Moreover, several genes related to pathogen responses are significantly upregulated upon ZIKV infection, but not perturbed in CMV-infected hNPCs. Conclusion: The presented study is the first to report enrichment of numerous pro-inflammatory pathways in ZIKV-infected hNPCs, indicating that hNPCs are capable of signaling through canonical pro-inflammatory pathways following viral infection. By defining gene expression profiles, new factors in the pathogenesis of ZIKV were identified which could help develop new therapeutic strategies. Show less

The single-stranded RNA Flavivirus, Zika virus (ZIKV), has recently re-emerged and spread rapidly across the western hemisphere's equatorial countries, primarily through Aedes mosquito transmission. While symptoms in adult infections appear to be self-limiting and mild, severe birth defects, such as microcephaly, have been linked to infection during early pregnancy. Recently, Tang et al. (Cell Stem Cell 2016, doi: 10.1016/j.stem.2016.02.016) demonstrated that ZIKV efficiently infects induced... Show moreThe single-stranded RNA Flavivirus, Zika virus (ZIKV), has recently re-emerged and spread rapidly across the western hemisphere's equatorial countries, primarily through Aedes mosquito transmission. While symptoms in adult infections appear to be self-limiting and mild, severe birth defects, such as microcephaly, have been linked to infection during early pregnancy. Recently, Tang et al. (Cell Stem Cell 2016, doi: 10.1016/j.stem.2016.02.016) demonstrated that ZIKV efficiently infects induced pluripotent stem cell (iPSC) derived human neural progenitor cells (hNPCs), resulting in cell cycle abnormalities and apoptosis. Consequently, hNPCs are a suggested ZIKV target. We analyzed the transcriptomic sequencing (RNA-seq) data (GEO: GSE78711) of ZIKV (Strain: MR766) infected hNPCs. For comparison to the ZIKV-infected hNPCs, the expression data from hNPCs infected with human cytomegalovirus (CMV) (Strain: AD169) was used (GEO: GSE35295). Utilizing a combination of Gene Ontology, database of human diseases, and pathway analysis, we generated a putative systemic model of infection supported by known molecular pathways of other highly related viruses. We analyzed RNA-sequencing data for transcript expression alterations in ZIKV-infected hNPCs, and then compared them to expression patterns of iPSC-derived hNPCs infected with CMV, a virus that can also induce severe congenital neurological defects in developing fetuses. We demonstrate for the first time that many of cellular pathways correlate with clinical pathologies following ZIKV infection such as microcephaly, congenital nervous system disorders and epilepsy. Furthermore, ZIKV activates several inflammatory signals within infected hNPCs that are implicated in innate and acquired immune responses, while CMV-infected hNPCs showed limited representation of these pathways. Moreover, several genes related to pathogen responses are significantly upregulated upon ZIKV infection, but not perturbed in CMV-infected hNPCs. The presented study is the first to report enrichment of numerous pro-inflammatory pathways in ZIKV-infected hNPCs, indicating that hNPCs are capable of signaling through canonical pro-inflammatory pathways following viral infection. By defining gene expression profiles, new factors in the pathogenesis of ZIKV were identified which could help develop new therapeutic strategies. Show less

Mutations in sarcomeric proteins have recently been established as heritable causes of Restrictive Cardiomyopathy (RCM). RCM is clinically characterized as a defect in cardiac diastolic function, such as, impaired ventricular relaxation, reduced diastolic volume and increased end-diastolic pressure. To date, mutations have been identified in the cardiac genes for desmin, alpha-actin, troponin I and troponin T. Functional studies in skinned muscle fibers reconstituted with troponin mutants... Show moreMutations in sarcomeric proteins have recently been established as heritable causes of Restrictive Cardiomyopathy (RCM). RCM is clinically characterized as a defect in cardiac diastolic function, such as, impaired ventricular relaxation, reduced diastolic volume and increased end-diastolic pressure. To date, mutations have been identified in the cardiac genes for desmin, alpha-actin, troponin I and troponin T. Functional studies in skinned muscle fibers reconstituted with troponin mutants have established phenotypes consistent with the clinical findings which include an increase in myofilament Ca(2+) sensitivity and basal force. Moreover, when RCM mutants are incorporated into reconstituted myofilaments, the ability to inhibit the ATPase activity is reduced. A majority of the mutations cluster in specific regions of cardiac troponin and appear to be mutational "hot spots". This paper highlights the functional and clinical characteristics of RCM linked mutations within the troponin complex. Show less

Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in... Show moreCircadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a beta-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca2+ level in cardiomyocytes using PER2:: LUC in combination with GCaMP3, a genetically encoded fluorescent Ca2+ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca2+ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca2+. Show less

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence... Show moreLatent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomalpackaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-kappa B and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen- activated protein kinase/ extracellular signal- regulated kinase (MAPK/ERK) and noncanonical NF-kappa B activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling. IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-kappa B and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis. Show less

Date Issued

2017-03

Identifier

FSU_libsubv1_wos_000394356400032, 10.1128/JVI.02251-16

Format

Citation

Title

Cdc45 protein-single-stranded DNA interaction is important for stalling the helicase during replication stress.

Creator

Bruck, Irina, Kaplan, Daniel L

Abstract/Description

Replicative polymerase stalling is coordinated with replicative helicase stalling in eukaryotes, but the mechanism underlying this coordination is not known. Cdc45 activates the Mcm2-7 helicase. We report here that Cdc45 from budding yeast binds tightly to long (≥ 40 nucleotides) genomic single-stranded DNA (ssDNA) and that 60mer ssDNA specifically disrupts the interaction between Cdc45 and Mcm2-7. We identified a mutant of Cdc45 that does not bind to ssDNA. When this mutant of cdc45 is... Show moreReplicative polymerase stalling is coordinated with replicative helicase stalling in eukaryotes, but the mechanism underlying this coordination is not known. Cdc45 activates the Mcm2-7 helicase. We report here that Cdc45 from budding yeast binds tightly to long (≥ 40 nucleotides) genomic single-stranded DNA (ssDNA) and that 60mer ssDNA specifically disrupts the interaction between Cdc45 and Mcm2-7. We identified a mutant of Cdc45 that does not bind to ssDNA. When this mutant of cdc45 is expressed in budding yeast cells exposed to hydroxyurea, cell growth is severely inhibited, and excess RPA accumulates at or near an origin. Chromatin immunoprecipitation suggests that helicase movement is uncoupled from polymerase movement for mutant cells exposed to hydroxyurea. These data suggest that Cdc45-ssDNA interaction is important for stalling the helicase during replication stress. Show less

Autosomal recessive primary microcephaly (MCPH) is characterized by small brain size as a result of deficient neuron production in the developing cerebral cortex. Although MCPH is a rare disease, the questions surrounding its etiology strike at the core of stem cell biology. The seven genes implicated in MCPH all encode centrosomal proteins and disruption of the MCPH gene Cdk5rap2 in mice revealed its role in neural progenitor proliferation and in maintaining normal centriole replication... Show moreAutosomal recessive primary microcephaly (MCPH) is characterized by small brain size as a result of deficient neuron production in the developing cerebral cortex. Although MCPH is a rare disease, the questions surrounding its etiology strike at the core of stem cell biology. The seven genes implicated in MCPH all encode centrosomal proteins and disruption of the MCPH gene Cdk5rap2 in mice revealed its role in neural progenitor proliferation and in maintaining normal centriole replication control. We discuss here the impact that centrosome regulation has upon neural progenitors in the developing brain. We integrate the impact of centriole replication defects with the functions of Cdk5rap2 and other MCPH proteins, propose mechanisms for progenitor loss in MCPH, and discuss links to two other microcephaly syndromes. Show less

Cocaine is a highly abused drug that causes psychiatric and neurological problems. Its entry into neurons could alter cell-biochemistry and contribute in the manifestation of early pathological symptoms. We have previously shown the acute cocaine effects in rat C6 astroglia-like cells and found that these cells were highly sensitive to cocaine in terms of manifesting certain pathologies known to underlie psychological disorders. The present study was aimed to discern acute cocaine effects on... Show moreCocaine is a highly abused drug that causes psychiatric and neurological problems. Its entry into neurons could alter cell-biochemistry and contribute in the manifestation of early pathological symptoms. We have previously shown the acute cocaine effects in rat C6 astroglia-like cells and found that these cells were highly sensitive to cocaine in terms of manifesting certain pathologies known to underlie psychological disorders. The present study was aimed to discern acute cocaine effects on the early onset of various changes in Neuro-2a (N2a) cells. Whole-cell patch-clamp recording of differentiated cells displayed the functional voltage-gated Na and K channels, which demonstrated the neuronal characteristics of the cells. Treatment of these cells with acute cocaine (1 h) at in vivo (nM to μM) and in vitro (mM) concentrations revealed that the cells remained almost 100% viable. Cocaine administration at 6.25 μM or 4 mM doses significantly reduced the inward currents but had no significant effect on outward currents, indicating the Na channel-blocking activity of cocaine. While no morphological change was observed at in vivo doses, treatment at in vitro doses altered the morphology, damaged the neurites, and induced cytoplasmic vacuoles; furthermore, general mitochondrial activity and membrane potential were significantly decreased. Mitochondrial dysfunction enabled the cells switch to anaerobic glycolysis, evidenced by dose-dependent increases in lactate and HS, resulting unaltered ATP level in the cells. Further investigation on the mechanism of action unfolded that the cell's resistance to cocaine was through the activation of nuclear factor E2-related factor-2 () gene and subsequent increase of antioxidants (glutathione [GSH], catalase and GSH peroxidase [GPx]). The data clearly indicate that the cells employed a detoxifying strategy against cocaine. On a broader perspective, we envision that extrapolating the knowledge of neuronal resistance to central nervous system (CNS) diseases could delay their onset or progression. Show less

The fragile X mental retardation protein (FMRP) plays an important role in normal brain development. Absence of FMRP results in abnormal neuronal morphologies in a selected manner throughout the brain, leading to intellectual deficits and sensory dysfunction in the fragile X syndrome (FXS). Despite FMRP importance for proper brain function, its overall expression pattern in the mammalian brain at the resolution of individual neuronal cell groups is not known. In this study we used FMR1... Show moreThe fragile X mental retardation protein (FMRP) plays an important role in normal brain development. Absence of FMRP results in abnormal neuronal morphologies in a selected manner throughout the brain, leading to intellectual deficits and sensory dysfunction in the fragile X syndrome (FXS). Despite FMRP importance for proper brain function, its overall expression pattern in the mammalian brain at the resolution of individual neuronal cell groups is not known. In this study we used FMR1 knockout and isogenic wildtype mice to systematically map the distribution of FMRP expression in the entire mouse brain. Using immunocytochemistry and cellular quantification analyses, we identified a large number of prominent cell groups expressing high levels of FMRP at the subcortical levels, in particular sensory and motor neurons in the brainstem and thalamus. In contrast, many cell groups in the midbrain and hypothalamus exhibit low FMRP levels. More important, we describe differential patterns of FMRP distribution in both cortical and subcortical brain regions. Almost all major brain areas contain high and low levels of FMRP cell groups adjacent to each other or between layers of the same cortical areas. These differential patterns indicate that FMRP expression appears to be specific to individual neuronal cell groups instead of being associated with all neurons in distinct brain regions, as previously considered. Taken together, these findings support the notion of FMRP differential neuronal regulation and strongly implicate the contribution of fundamental sensory and motor processing at subcortical levels to FXS pathology. Show less

The centrosome is the best-understood microtubule-organizing center (MTOC) and is essential in particular cell types and at specific stages during development. The centrosome is not required zygotically for mitosis or to achieve full animal development. Nevertheless, centrosomes are essential maternally during cleavage cycles in the early embryo, for male meiotic divisions, for efficient division of epithelial cells in the imaginal wing disc, and for cilium/flagellum assembly in sensory... Show moreThe centrosome is the best-understood microtubule-organizing center (MTOC) and is essential in particular cell types and at specific stages during development. The centrosome is not required zygotically for mitosis or to achieve full animal development. Nevertheless, centrosomes are essential maternally during cleavage cycles in the early embryo, for male meiotic divisions, for efficient division of epithelial cells in the imaginal wing disc, and for cilium/flagellum assembly in sensory neurons and spermatozoa. Importantly, asymmetric and polarized division of stem cells is regulated by centrosomes and by the asymmetric regulation of their microtubule (MT) assembly activity. More recently, the components and functions of a variety of non-centrosomal microtubule-organizing centers (ncMTOCs) have begun to be elucidated. Throughout development, a wide variety of unique ncMTOCs form in epithelial and non-epithelial cell types at an assortment of subcellular locations. Some of these cell types also utilize the centrosomal MTOC, while others rely exclusively on ncMTOCs. The impressive variety of ncMTOCs being discovered provides novel insight into the diverse functions of MTOCs in cells and tissues. This review highlights our current knowledge of the composition, assembly, and functional roles of centrosomal and non-centrosomal MTOCs in . Show less

Characterization of binding of LARP6 to the 5' stem-loop of collagen mRNAs: implications for synthesis of type I collagen..

Creator

Stefanovic, Lela, Longo, Liam, Zhang, Yujie, Stefanovic, Branko

Abstract/Description

Type I collagen is composed of 2 polypeptides, α1(I) and α2(I), which fold into triple helix. Collagen α1(I) and α2(I) mRNAs have a conserved stem-loop structure in their 5' UTRs, the 5'SL. LARP6 binds the 5'SL to regulate type I collagen expression. We show that 5 nucleotides within the single stranded regions of 5'SL contribute to the high affinity of LARP6 binding. Mutation of individual nucleotides abolishes the binding in gel mobility shift assay. LARP6 binding to 5'SL of collagen α2(I)... Show moreType I collagen is composed of 2 polypeptides, α1(I) and α2(I), which fold into triple helix. Collagen α1(I) and α2(I) mRNAs have a conserved stem-loop structure in their 5' UTRs, the 5'SL. LARP6 binds the 5'SL to regulate type I collagen expression. We show that 5 nucleotides within the single stranded regions of 5'SL contribute to the high affinity of LARP6 binding. Mutation of individual nucleotides abolishes the binding in gel mobility shift assay. LARP6 binding to 5'SL of collagen α2(I) mRNA is more stable than the binding to 5'SL of α1(I) mRNA, although the equilibrium binding constants are similar. The more stable binding to α2(I) mRNA may favor synthesis of the heterotrimeric type I collagen. LARP6 needs 2 domains to contact 5'SL, the La domain and the RRM. T133 in the La domain is critical for folding of the protein, while loop 3 in the RRM is critical for binding 5'SL. Loop 3 is also involved in the interaction of LARP6 and protein translocation channel SEC61. This interaction is essential for type I collagen synthesis, because LARP6 mutant which binds 5'SL but which does not interact with SEC61, suppresses collagen synthesis in a dominant negative manner. We postulate that LARP6 directly targets collagen mRNAs to the SEC61 translocons to facilitate coordinated translation of the 2 collagen mRNAs. The unique sequences of LARP6 identified in this work may have evolved to enable its role in type I collagen biosynthesis. Show less

Neuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified... Show moreNeuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified from Escherichia coli BL21(DE3) cells. We found that all three types of n-Src constructs expressed at very high yields (∼500 mg/L) at 37°C, but formed inclusion bodies. In the presence of 8M urea these proteins could be solubilized, purified under denaturing conditions, and subsequently refolded in the presence of arginine (0.5M). These Src proteins were enzymatically active except for the n-Src/K303R/Y535F mutant. n-Src proteins expressed at 18°C were soluble, albeit at lower yields (∼10-20 mg/L). The lowest yields were for n-Src/Y535F (∼10 mg/L) and the highest for n-Src/K303R/Y535F (∼20 mg/L). We characterized the purified n-Src proteins expressed at 18°C. We found that altering n-Src enzyme activity either pharmacologically (e.g., application of ATP or a Src inhibitor) or genetically (mutation of Y535 or K303) was consistently associated with changes in n-Src stability: an increase in n-Src activity was coupled with a decrease in n-Src stability and vice versa. These findings, therefore, indicate that n-Src activity and stability are interdependent. Finally, the successful production of functionally active n-Src in this study indicates that the bacterial expression system may be a useful protein source in future investigations of n-Src regulation and function. Show less

Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds KRS, impinged on the interaction of KRS with 67LR and suppressed metastasis in three different mouse models. The compound inhibited the... Show moreLysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds KRS, impinged on the interaction of KRS with 67LR and suppressed metastasis in three different mouse models. The compound inhibited the KRS-67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS-67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS. Show less